Week 1

Sporulation salts were made and used to make sporulation plates. Gridded microscopy plates were prepared using a wax pencil. Dead Varroa mites were gathered from the Unifarm location at Grebbedijk, Wageningen. These beehives were not treated against mites and contained a high number of them. The mites were not completely fresh.

Week 2

It was not known how many colonies could be isolated from one mite; therefore, microcentrifuge tubes were prepared with 1, 2, or 3 mites, 2 replicates each. The protocol for Bacillus isolation was followed, except the sterilization step was with 1% halamid solution and lasted 10 seconds.

Initial plates showed no colonies; the experiment was repeated with a ~2.5% bleach washing step.

The second set of plates also showed no colonies. After the heat treatment, the LB + mites was incubated for 24 hours and streaked with an inoculation loop.

Again, no colonies: a mite was dissected and its gut rubbed on a plate. Due to the small size of the mite, it was difficult to isolate the gut. Additionally, the same protocol as before was followed, except only 0.1 mL of the LB was heat treated and the rest was plated without a heat treatment.

This week, the protocol failed to yield any colonies. I also tried to plate mite samples without a sterilization step. During dissection of the dead mite, I was able to observe dessication; this indicated that the mites were too dry to avoid sucking up bleach during the sterilization step. Their guts were probably also sterile, which explains the lack of colonies.

To test this hypothesis, I repeated the isolation protocol without a sterilization step and only washed with sterile tap water. This yielded 3 colonies, but now I would be unable to exclude environmental contamination.

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 16

Week 17

Week 18

Week 19

Week 8

Week 20

Week 21

Week 22

Week 23